mobilization after compression of the median nerve 夽

SOCIEDADE BRASILEIRA DE ORTOPEDIA E TRAUMATOLOGIA

w w w . r b o . o r g . b r

Original article

Neurotrophin expression and histomorphometric evaluation in Wistar rats subjected to neural

mobilization after compression of the median nerve ^夽

Marieli Araujo Rossoni Marcioli

, José Luis da Conceic¸ão Silva

, Lucinéia de Fátima Chasko Ribeiro

, Rose Meire Costa Brancalhão

, Gladson Ricardo Flor Bertolini

aUniversidadeEstadualdoOestedoParaná(Unioeste),Cascavel,PR,Brazil

bProgramadeCiênciasFarmacêuticas,UniversidadeEstadualdoOestedoParaná(Unioeste),Cascavel,PR,Brazil

cProgramadeBiociênciaseSaúde,UniversidadeEstadualdoOestedoParaná(Unioeste),Cascavel,PR,Brazil

a r t i c l e i n f o

Articlehistory:

Received16January2017 Accepted16March2017 Availableonline4April2018

Keywords:

Nerveregeneration Histology

Intercellularsignalingpeptidesand proteins

Exercise Mediannerve

a bs t r a c t

Objective:ToevaluatetheneurotrophinmRNAexpressionandaxoncountinthemedian nerveofWistarratssubmittedtoneuralmobilization(NM)afternervecompression.

Methods:EighteenanimalswererandomlydividedintoG1(nervecompressiononly),G2 (NMfor1min),andG3(NMfor3min).ForNM,theanimalswereanesthetizedandtheright scapulareceivedthemobilization,adaptedasindicatedforhumans,onalternatedays,from thethirdtothe13thpostoperative(PO)day,totalingsixdaysoftherapy.Onthe14thPOday, animalswereanesthetizedandeuthanized.Fragmentsofthemediannerve,distaltothe compressionprocedure,wereremovedforhistomorphometricanalysisandexpressionof neurotrophins,nervegrowthfactor(NGF),andbrain-derivedneurotrophicfactor(BDNF)by RT-PCR.

Results:Histomorphometricanalysisrevealeddifferencesinthenumberofaxonsinthe injuredside,whichwassignificantlylowerintheinjuredlimbnervecomparedtothecontrol limb,whereastheRT-PCRanalysisshowednosignificantdifferencesintheexpressionof NGForBDNF.

Conclusion:NMtreatmentdidnotaffectmediannerveregeneration,whichmaintainednor- malrecoveryrates.

©2017SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

夽StudyconductedatUniversidadeEstadualdoOestedoParaná(Unioeste),Cascavel,PR,Brazil.

∗ Correspondingauthor.

E-mail:gladsonricardo@gmail.com(G.R.Bertolini).

https://doi.org/10.1016/j.rboe.2018.03.006

2255-4971/©2017SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditoraLtda.Thisisanopenaccessarticle undertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Expressãodeneurotrofinaseavaliac¸ãohistomorfométricaemratos Wistarsubmetidosàmobilizac¸ãoneuralapóscompressãodonervo mediano

Palavras-chave:

Regenerac¸ãonervosa Histologia

Peptídeoseproteínasde sinalizac¸ãointercelular Exercício

Nervomediano

r e s u mo

Objetivo: AvaliaraexpressãodeRNAmdeneurotrofinaseacontagemdeaxôniosnonervo medianoderatosWistarsubmetidosàmobilizac¸ãoneural(MN)apóscompressãonervosa.

Métodos: Foramdivididosaleatoriamente18animaisemG1(apenascompressãonervosa), G2(MNpor1minuto)eG3(MNpor3minutos).ParaaMN,osanimaisforamanestesia- doseomembroescapulardireitorecebeuamobilizac¸ão,adaptadadaformaindicadapara humanos,emdiasalternados,doterceiroao13^◦diadepós-operatório(PO),emseisdiasde terapia.No14^◦diaPO,osanimaisforamanestesiadoseeutanasiados.Fragmentosdonervo mediano,distaisaoprocedimentodecompressão,foramretiradosparaanálisehistomor- fométricaedeexpressãodasneutrotrofinas,fatordecrescimentodonervo(NGF)efatorde crescimentoderivadodocérebro(BNDF)porRT-PCR.

Resultados: Aanálisehistomorfométricaevidencioudiferenc¸asnonúmerodeaxôniosnos ladoslesionados,quefoisignificativamentemenornonervodomembrolesadocomparado comomembrocontrole;porsuavez,aanáliseporRT-PCRnãoapontoudiferenc¸assigni- ficativasnaexpressãodeNGFenemdeBNDF.

Conclusão: OtratamentodeMNnãoafetouaregenerac¸ãodonervomediano,quemanteve índicesnormaisderecuperac¸ão.

©2017SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http://

creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Carpaltunnelsyndrome(CTS)isthemostcommonperipheral nervecompression;itsincidenceishigh,beingmorepreva- lentinwomenandtheelderly.Itpresentsasacompression ofthe mediannerve when it passes under the transverse carpal ligament,¹ which initially causes blockage of the venousflowfollowedbyaninflammatoryreaction, fibrosis, demyelination, and axonal loss. In addition, an increase inthe expressionofprostaglandin E2,vascular endothelial growthfactor,andinterleukin6isobserved.Theclinicalsigns andsymptomsofCTSarenumbnessandparesthesiainthe firstthreefingersandintheradialsideofthefourthfinger, togetherwithnocturnalarousalduetopain,besidesmuscle weakness,andatrophy.²

Carpal tunnel decompression surgery is performed in severeandrefractorycases.Conservativetreatmentisused inmildandmoderateones¹andpresentsasadvantagesthe reduced number ofcomplicationsand its lower cost. Non- surgicaltreatmentincludetheuseofasplint,non-hormonal anti-inflammatory drugs, local corticosteroid injections, in additiontophysicaltherapeuticresourcessuchasultrasound, low-powerlaser,andexercise.²

TheEuropeanGuidelineforthetreatmentofCTSindicates thattheapproachmustbemultidisciplinary,andthatthera- peuticexercisessuchasneuralmobilization(NM)canbecon- sideredatreatmentoption.³Thetechniquehasbeenshownto producechangesintheperipheralnerveexcursion⁴thatcan preventorreduceintraneuraledemaand,thus,rehabilitate normalnervefunction.⁵ Althoughitisalready widelyused

amongphysicaltherapists,theliteraturedoesnotyetprovide evidenceofitspositiverepercussioninthetreatmentofCTS.⁶ Wallerian degeneration is observed in cases of severe nervecompression,leadingtomorphologicalalterations⁷and subsequentproductionofneurotrophins.⁸Neurotrophicfac- tors (such as nerve growth factor [NGF] and brain-derived neurotrophicfactor[BDNF])regulateneuron survival,devel- opment,differentiation,andregeneration.Theyarereleased bySchwanncellsafteraxonotmesis,andcompleteneuronal regenerationdependsontheirexpressionandtrophiccondi- tionsfavorabletoproteinsynthesis.⁹

Consideringsomecontroversiesregardingtheefficacyof NMdirectlyonthemediannerve,thepresentstudychecked its effects on the number of axons and in the expression ofneurotrophins(NGFand BDNF)intwodifferenttimesof applicationofthetechniqueinratssubmittedtoanervecom- pressionmodel.

Material and methods

Thepresentresearchwasexperimental.Theprojectwascon- ductedaccordingtotheinternationalguidelinesofresearch ethicsinanimalstudiesandapprovedbytheCommitteeof Ethics in Animal Use (opinion No 1012/12). Eighteen male Wistarrats,14±2weeksold,werekeptona12hphotoperiod, 23±1^◦C,withwaterand foodadlibitum.Theanimalswere randomlydividedintothreegroups:G1(n=6)–submittedto neuralcompressionwithouttreatment(onlyanesthetized);G2 (n=6)–submittedtoneuralcompressionandtreatedwithNM for1min;G3(n=6)–submitted toneuralcompression and treatedwithNMfor3min.

Mediannervecompression

ThemodelpresentedbyChenetal.¹⁰ wasusedformedian nervecompression, whichwas madewithchromed Catgut 4.0suturelinein4points,withanapproximatedistanceof 1mm,onthemedialnerve,intheproximalregionoftheright elbow.Forthesurgicalnervecompressionprocedure,theani- malswerepreviouslyanesthetizedwithaketamine(50mg/kg) andxylazine(10mg/kg)solution.

Mediannervemobilization

For NM (G2 and G3), the animals were anesthetized and the treatment was performed on the right scapular limb, asadaptedfromthetreatmentinhumans.Theanimalwas placedindorsalrecumbentposition,withlateralcervicalflex- iontotheleft,depressionoftheshouldergirdleand slight abduction,externalrotationandsupination,withmaximum extensionpossibleattheelbowandwristuntilaresistance tomovementwasobserved.Inthisposition,repeatedoscil- lationsofwristflexion-extensionsweremadefor1min(G2) or3min(G3).Sixsessionswereperformedonalternatedays betweenthethirdand13thpostoperative(PO)days.¹¹Onthe samedaysoftreatmentofG2andG3,theG1animalswere anesthetizedandplacedbackintotheirboxes,withoutunder- goingtheprotocol.

Histomorphometricassessment

Attheendofthetreatmentperiod,onthe14thPOday,the animalswereanesthetizedandtwofragmentsofthemedian nerveweredissectedandremovedfromtheregiondistalto thecompressionprocedure.Anervefragmentofthecontralat- erallimbwasalsoremovedforcomparisonwiththeinjured side;theanimalswerelatereuthanizedbydecapitation.The fragmentswerefixedin10%formalinandprocessedforinclu- sioninhistologicalparaffin.Thematerialwascross-sectioned into 5␮m- thick slices, and stained with hematoxylin andeosin.

Imagesoffourvisualfieldswerecapturedintheupperleft and rightand lower right and left quadrantsusing a100× lens,accordingtotherecommendationsofGeunaetal.¹²The imageswereanalyzedwiththeImage-Pro-Plus6.0software, inwhichthenumberofaxonsineachquadrantwascounted, inanareaof23,184␮m².Theanimalsweresubdividedinto thosethatpresentednormalitycharacteristics(withthepres- enceofmyelinsheathand axon)andthose thatpresented degenerationcharacteristics.

ExpressionofNGFandBDNFneurotrophins

Thedistalnervefragments, storedat−80^◦C,were homog- enized to isolate the total RNA, using the Trizol reagent (Invitrogen) according to the manufacturer’s recommen- dations. The total extracted RNA was dissolved in water treated with diethylpyrocarbonate (DEPC), quantified at 260/280nm in a spectrophotometer (Genesys 10S UV–VIS, ThermoScientific, USA)and subjected tothe reverse tran- scription procedure by RT-PCRtechnique, according to the schemeshown below.ThecDNAwas synthesizedwiththe

enzyme Superscript II (Invitrogen) according to the manu- facturer’sprotocol.AftercDNAsynthesis,PCRamplification was performed; PCR reactions were performed with 2␮l cDNAwiththeprimersG3PDH(control–T^◦C53^◦C),NGF(T^◦C 60^◦C),andBDNF(T^◦C53^◦C).Theannealingtemperaturesin parentheseswerespecificfortheprimers.Atotalof35PCR cycles(94^◦C,annealingtemperature:72^◦C)wereperformed.

G3PDHwasconsideredacontrolgene.Thefollowingprimer pairs were used: BDNF: 5AATCCCATGGGTTACACGAA3

and 5AAGTTGTGCGCAAATGACTG3;

NGF: 5ATCCACCCACCCAGTCTTCCACAT3

and 5GGCAGCCTGTTTGTCGTCTGTTGT3;

GAPDH: 5CCTCTGGAAAGCTGTGGCGT3 and

5TTGGAGGCCATGTAGGCCAT3

PCR products were separated by 1% agarose gel elec- trophoresisandvisualizedbyanultraviolettransilluminator (UVP-USA).

Dataanalysis

Theresultswereexpressedandanalyzedthroughthedescrip- tiveandinferentialstatistics,usingtheBioestat5.0software.

Factorialanalysisofvariance(ANOVA)wasused,andtheright andleftsideswereconsideredasblocks;thelevelofsignifi- cancewassetat5%.

Results

Regarding the axon count, no difference was observed betweenthetreatments(F=2.97,p=0.06),thoughdifferences wereobservedbetweentheinjuredandcontralateralnerves (F=9.31,p=0.004),without interaction(F=0.81,p=0.54). For thoseanimalsthatpresenteddegeneration,theresultswere similar, with no differences between the groups (F=2.13, p=0.13),whiledifferenceswereobservedbetweenthesides (F=6.25, p=0.01), but without interaction (F=2.68, p=0.08;

Table1).

RegardingthegeneexpressionofNGFandBDNF,nodiffer- encewasobservedbetweenthegroups(Fig.1).G3PDH(control) geneamplificationwasobserved,demonstratingtheconstitu- tiveexpressionofthisgene;however,theNGFandBDNFgenes werenotexpressedinamountsdetectableintheexperiment.

Table1–Resultsofthehistomorphometricanalysisof theright(R)andleft(L)mediannerve,inrelationtothe axoncount.

Right(injured)^a Left(control)^a

Normal

G1 47.21±44.95 66.50±50.92

G2 73.58±57.05 129.8±34.36

G3 40.96±56.92 104.9±15.11

Degenerated

G1 8.00±12.43 3.62±8.88

G2 5.17±11.36 0±0

G3 31.96±35.85 0±0

a Significantdifferenceswereobservedbetweensides.

M

600 pb

1C 1B 1N 6C 6B 6N M

Fig.1–AnalysisoftheproductsamplifiedbyRT-PCRin1%

agarosegelelectrophoresis.M=DNAladder100pb;1Cand 6CcDNAamplifiedfortheG3PDHcontrolgene(amplified bandof430bp).1Band6B:cDNAamplifiedwiththeBDNF primersand1Nand6N:cDNAamplifiedwiththeNGF primers.gr1.

AweakamplificationoftheBDNFgenewasobservedingroup 1B(385bpamplifiedband).

Discussion

AlthoughNMisusedtogainflexibility,^13,14musclestrength,¹⁵ reduce pain,^16–19 and accelerate tissue repair,¹¹ there are controversiesregardingtheefficacyofthistechniqueinexper- imentalstudiesinindividuals withnervecompression;this mightbeduetothediversityofprotocolsused,withdifferent intensitiesandpositioning.⁴Thepresentstudyalsopresented a different protocol, which sought to adapt, in an animal model,the positioning ofmediannervestretching done in clinicalpracticewithhumans.²⁰

Thehypothesisonthebenefitsofthetechniquearethatit facilitatesnervegliding,reducesadherences,dispersesnox- iousfluids, increases neural vascularization, and improves axoplasmaticflow.However,thesemechanismsstillrequire validation.²¹ According toGinanneschiet al.,²² the tension produced iscapableofreducing nerve conduction failures;

cautionshouldbeexercisedwhenusingsuchtechniquesin patientswithCTS.

Regardless of the time span of1 or 3min, NM did not presentdifferencesinrelationtothecontrolgroup;smaller values of cells with normal characteristics and a greater amountofcellsindegenerationwereobserved.Thesefindings wereconsistentwithapreviousstudy,inwhichnoimprove- mentwasobservedwithNMinthisexperimentalmodelof injury,invariablessuchasnervefibersand axondiameter, myelinsheaththickness,myelin/axonratio,andGquotient, aswellastherewasnonociceptivereduction.¹¹Despitethe lackofimprovementinsuchparameters,itwasbelievedthat themechanicalstimulusappliedwiththeMNtechniquecould stimulatethenervebudding²³andthusincreasethenumber ofnervefibers,aswellastheproductionofneurotrophinswith theaimofrepairingnervoustissue.²⁴

Santosetal.²⁵alsousednervecompressionbytyingsuture lines, but in the sciatic nerve ofrats, and performed NM 14 daysafter the injuryprotocol onalternate days forten

sessions; those authors observed functional improvement of treated animals, recovery of the anterior tibial muscle strength, and increasednumber of opioid-kappareceptors in the periaqueductal gray matter. In another study, the authors²⁶observedthatthetreatedanimalspresentedlower nociception (bothallodynia and hyperalgesia)and reduced levelsofNGFinthedorsalrootgangliaipsilateraltothelesion, whichcouldbeapossiblereasonforthereductioninpain.

Nonetheless,anotherstudy fromthesamegroup²⁷ indi- cated that neural mobilization produced some recovery characteristics,suchasagreaternumberofaxonswithnormal thicknessmyelinsheathsandlessadherencesbetweenaxons, but locallytherewasanincreaseintheNGFconcentration evaluatedbyWesterblotting.

Inthepresent study,theauthorsalsosoughttoanalyze theexpressionofneurotrophinsatthecompressionsite;how- ever,unliketheabove-mentionedauthors,nodifferenceswere observedintheirexpressionwiththeuseoftheRT-PCRtech- nique.Itshouldbenotedthatneuralmobilizationwasused early,thatis,inthefirsttwoweeksaftercompressionthrough suturelines;itwouldbepossibletodelaytreatmentonsetand itcouldbebeneficial.Thisisanotherlimitationofthestudy andapossiblethemeforfuturestudies.

Conclusion

Withtheprotocolsused,NMdidnotfavornerveregeneration inamediannervecompressionmodelofWistarrats,which maintainednormalrecoveryrates.

Conflicts of interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TothePhysicalRehabilitationCenterofUnioesteforthepartial fundingofthisresearch.

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